U.S. patent number 8,877,199 [Application Number 13/320,630] was granted by the patent office on 2014-11-04 for b cell surface reactive antibodies.
This patent grant is currently assigned to The United States of America, as represented by the Secretary of the Department of Health and Human Services. The grantee listed for this patent is Sivasubramanian Baskar, Michael R. Bishop, Christoph Rader, Ivan Samija, Jessica M. Suschak. Invention is credited to Sivasubramanian Baskar, Michael R. Bishop, Christoph Rader, Ivan Samija, Jessica M. Suschak.
United States Patent |
8,877,199 |
Rader , et al. |
November 4, 2014 |
B cell surface reactive antibodies
Abstract
The invention relates to antibodies that are reactive to the
cell surface of CD19+ B cells, including B-cell chronic lymphocytic
leukemia (B-CLL) cells, and compositions and methods for using such
antibodies, including in the diagnosis and treatment of disorders
associated with CD19+ B cells, such as B-CLL.
Inventors: |
Rader; Christoph (Olney,
MD), Baskar; Sivasubramanian (Elicott City, MD), Bishop;
Michael R. (Williams Bay, WI), Samija; Ivan (Zagreb,
HR), Suschak; Jessica M. (Jefferson, MA) |
Applicant: |
Name |
City |
State |
Country |
Type |
Rader; Christoph
Baskar; Sivasubramanian
Bishop; Michael R.
Samija; Ivan
Suschak; Jessica M. |
Olney
Elicott City
Williams Bay
Zagreb
Jefferson |
MD
MD
WI
N/A
MA |
US
US
US
HR
US |
|
|
Assignee: |
The United States of America, as
represented by the Secretary of the Department of Health and Human
Services (Washington, DC)
|
Family
ID: |
42537769 |
Appl.
No.: |
13/320,630 |
Filed: |
May 12, 2010 |
PCT
Filed: |
May 12, 2010 |
PCT No.: |
PCT/US2010/034491 |
371(c)(1),(2),(4) Date: |
February 01, 2012 |
PCT
Pub. No.: |
WO2010/132532 |
PCT
Pub. Date: |
November 18, 2010 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20120121504 A1 |
May 17, 2012 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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61178688 |
May 15, 2009 |
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Current U.S.
Class: |
424/155.1;
530/391.3; 530/388.8; 424/178.1; 424/136.1; 424/135.1; 530/388.15;
530/391.7; 424/142.1; 530/387.3 |
Current CPC
Class: |
G01N
33/56972 (20130101); C07K 16/005 (20130101); C07K
16/18 (20130101); C07K 16/3061 (20130101); A61P
35/02 (20180101); G01N 33/57426 (20130101); A61K
2039/505 (20130101); C07K 2317/56 (20130101); C07K
2317/50 (20130101); C07K 2317/55 (20130101); C07K
2317/21 (20130101); C07K 2317/565 (20130101) |
Current International
Class: |
A61K
39/395 (20060101); A61K 51/10 (20060101); C07K
16/30 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO2004/067569 |
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Aug 2004 |
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WO |
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WO 2004/067569 |
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Aug 2004 |
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WO |
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WO 2004/110369 |
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Dec 2004 |
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WO |
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WO 2005/030124 |
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Apr 2005 |
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WO |
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WO 2005/100605 |
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Oct 2005 |
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WO |
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WO 2007/141278 |
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Dec 2007 |
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WO |
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WO 2008/074004 |
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Jun 2008 |
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WO |
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WO 2008/103849 |
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Aug 2008 |
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WO |
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WO 2008/122039 |
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Oct 2008 |
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WO |
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WO 2009/018411 |
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Feb 2009 |
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WO |
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WO 2010/017103 |
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Feb 2010 |
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WO |
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|
Primary Examiner: Yu; Misook
Assistant Examiner: Allen; Michael D
Attorney, Agent or Firm: Leydig, Voit & Mayer, Ltd.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This patent application is a U.S. national phase of International
Patent Application No. PCT/US2010/034491, filed May 12, 2010, which
claims the benefit of U.S. Provisional Patent Application No.
61/178,688 filed May 15, 2009, the complete contents of which are
hereby incorporated by reference in their entireties.
Claims
The invention claimed is:
1. An isolated antibody having B-cell chronic lymphocytic leukemia
(B-CLL) cell surface reactivity, comprising: (i) the light chain
variable domain sequence of SEQ ID NO:1 and the heavy chain
variable domain sequence of SEQ ID NO:18; (ii) the light chain
variable domain sequence of SEQ ID NO:9 and the heavy chain
variable domain sequence of SEQ ID NO:26; (iii) the light chain
variable domain sequence SEQ ID NO:12 and the heavy chain variable
domain sequence of SEQ ID NO:31; or (iv) the light chain variable
domain sequence of SEQ ID NO:14 and the heavy chain variable domain
sequence of SEQ ID NO:33.
2. An isolated antibody with B-CLL cell surface reactivity,
comprising the following complementarity determining regions
(CDRs): (i) SEQ ID NO:3 as CDRL1, SEQ ID NO:5 as CDRL2, SEQ ID NO:7
as CDRL3, SEQ ID NO:20 as CDRH1, SEQ ID NO:22 as CDRH2, and SEQ ID
NO:24 as CDRH3; (ii) SEQ ID NO:3 as CDRL1, SEQ ID NO:5 as CDRL2,
SEQ ID NO:7 as CDRL3, SEQ ID NO:28 as CDRH1, SEQ ID NO:30 as CDRH2,
and SEQ ID NO:24 as CDRH3; or (iii) SEQ ID NO:3 as CDRL1, SEQ ID
NO:16 as CDRL2, SEQ ID NO:7 as CDRL3, SEQ ID NO:28 as CDRH1, SEQ ID
NO:30 as CDRH2, and SEQ ID NO:24 as CDRH3.
3. The antibody of claim 2, wherein the antibody includes SEQ ID
NO:23 as VH framework region 3, SEQ ID NO:24 as CDRH3, AND SEQ ID
NO:25 as VH framework region 4.
4. The antibody of claim 2, wherein the antibody includes SEQ ID
NO:3 as CDRL1, SEQ ID NO:4 as VL framework region 2, SEQ ID NO:7 as
CDRL3, and SEQ ID NO:8 as VL framework region 4.
5. The antibody of claim 2, wherein the antibody is selected from
the group consisting of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3,
IgG4, IgM, F(ab)2, Fv, scFv, IgG.DELTA.CH.sub.2, F(ab')2, Fab,
dsFv, Fv, scFv-Fc, a non-depleting IgG, a diabody, a T-body, a
bispecific antibody, and a bivalent antibody.
6. The antibody of claim 5, wherein the antibody is an IgG selected
from the group consisting of IgG1, IgG2, IgG3, IgG4, and synthetic
IgG.
7. The antibody of claim 5, wherein the antibody is a Fab.
8. The antibody of claim 5, wherein the antibody is a dsFv.
9. The antibody of claim 2, wherein the antibody is conjugated to a
synthetic molecule.
10. The antibody of claim 9, wherein the antibody is a T-body, and
the synthetic molecule comprises a transmembrane region and an
intracellular T-cell receptor (TCR) signaling domain.
11. The antibody of claim 9, wherein the synthetic molecule is a
label.
12. The antibody of claim 9, wherein the synthetic molecule is a
cytotoxic agent or a therapeutic radioisotope.
13. The antibody of claim 2, wherein the antibody is a fully human
antibody.
14. A pharmaceutical composition comprising a therapeutically
effective amount of an isolated antibody of claim 2 and a
pharmaceutically acceptable carrier.
15. A kit comprising the isolated antibody of claim 2.
16. The kit of claim 15, further comprising one or more immunoassay
buffers.
17. The kit of claim 15, wherein the antibody is conjugated to a
label.
Description
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY
Incorporated by reference in its entirety herein is a
computer-readable nucleotide/amino acid sequence listing submitted
concurrently herewith and identified as follows: One 17,116 Byte
ASCII (Text) file named "709042 ST25.TXT," created on Oct. 11,
2011.
BACKGROUND OF THE INVENTION
Antibody therapies and diagnostics have been developed for use in
treating a wide range of conditions including autoimmune diseases
or disorders, infectious diseases, and cancers. Such therapies are
useful but also can be undesirably immunogenic and damaging to
healthy cells and tissues.
About 30% of all diagnosed leukemias are B-cell chronic lymphocytic
leukemia (B-CLL), and the incidence of B-CLL is estimated to
include 15,000 new cases and 4,500 deaths in the United States
alone. Although generally considered incurable, a number of
chemotherapies and biological therapies have been clinically tested
in B-CLL patients. Of these, allogeneic hematopoietic stem cell
transplantation (alloHSCT) may be the only curative treatment for
some B-CLL patients. Boyiadzis et al., Expert Opin. Biol. Ther., 7:
1789-1797 (2007) and Gribben, J. G., Biol. Blood Marrow Transplant,
15: 53-58 (2008). AlloHSCT is believed to induce a
graft-versus-leukemia (GVL) response in alloHSCT recipients. This
GVL may be mediated by alloreactive donor T cells and/or donor B
cell-derived allo-HSCT-induced antibodies. Bleakley et al., Nat.
Rev. Cancer, 4: 371-380 (2004), Hambach et al., Curr. Opin.
Immunol., 17: 202-210 (2005), and Wu et al., Adv. Immunol., 90:
133-173 (2006). Other clinically tested biological therapies
include the use of rituximab and alemtuzumab. These therapeutic
antibodies, however, target antigens found on both malignant and
normal B cell surfaces. The CD52 antigen targeted by alemtuzumab is
also expressed on the cell surface of a variety of other normal
immune system cells. Accordingly, immunosupression can be a concern
with such antibodies.
Strategies that have been used in attempts to identify antigens
specific to malignant B-cells include differential gene expression
profiling and SEREX analysis. SEREX involves using serum antibodies
from cancer patients to screen recombinant cDNA expression
libraries. These strategies, however, do not necessarily
distinguish intracellular antigens from cell surface antigens.
There is a desire for additional antibody therapies that
preferentially target malignant B cell surface antigens, have good
efficacy, and are minimally immunogenic and/or damaging to
non-diseased cells.
BRIEF SUMMARY OF THE INVENTION
The invention provides an isolated antibody with specificity for a
cell surface marker found on certain CD19+ B cells such as B-CLL.
In particular, the invention provides an isolated antibody that has
B-CLL cell surface reactivity and includes (a) a light chain
variable domain having at least 90% identity to a sequence selected
from the group consisting of SEQ ID NO:1, SEQ ID NO:9, SEQ ID
NO:12, and SEQ ID NO:14, (b) a heavy chain variable domain having
at least 90% identity to a sequence selected from the group
consisting of SEQ ID NO:18, SEQ ID NO:26, SEQ ID NO:31, and SEQ ID
NO:33, or (c) both a light chain of (a) and a heavy chain of
(b).
The invention also provides an isolated antibody that has B-CLL
cell surface reactivity and includes at least one complementarity
determining region (CDR) having a sequence selected from the group
consisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:16,
SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:28, and SEQ ID
NO:30. In other embodiments, the isolated antibody with B-CLL cell
surface reactivity can include one or more variants of the
foregoing CDRs which have 1, 2, or 3 amino acid substitutions,
insertions, or deletions.
The invention further provides a pharmaceutical composition
comprising an antibody of the invention and a pharmaceutically
acceptable carrier.
Additionally, the invention provides a method of treating a disease
or condition, such as B-CLL, which is associated with elevated
levels of CD19+ B cells that are reactive with the antibody of the
invention. Generally the method of treatment includes administering
a therapeutically effective amount of an isolated antibody of the
invention or a pharmaceutical composition thereof to a subject in
need thereof.
The antibodies and compositions of the invention can also be used
in diagnostic methods to detect altered levels of CD19+ B cells
that are reactive with the antibody of the invention, e.g., B-CLL
cells, in a sample or in a subject.
In yet another embodiment, the invention provides an isolated B-CLL
cell surface marker that is reactive with the antibody of the
invention.
BRIEF DESCRIPTION OF THE DRAWING(S)
FIG. 1 is a schematic diagram that depicts cell surface markers on
B-CLL, including surface immunoglobulin (Ig), CD20, CD32B, and
other Fcy receptors.
FIG. 2 is a series of three histogram panels labeled A, B, and C,
respectively, depicting the results (in terms of the number of
events versus fluorescence intensity) of flow cytometry studies
including B-CLL and goat F(ab').sub.2 anti-human IgG polyclonal
antibodies conjugated to Qdot 655 nanocrystals ("g.alpha.h-Qdot")
(panel A), blocked B-CLL and g.alpha.h-Qdot (panel B), or blocked
B-CLL, rituximab, and g.alpha.h-Qdot (panel C).
FIG. 3A is a graph depicting the results of a flow cytometry study
of serum plasma reactivity to B-CLL cells (CD3-, CD19+) or T cells
(CD3+, CD19-); serum plasma was obtained from Patient A at the
indicated periods before (pre-), during (peri-), or after (post-)
alloHSCT, and flow cytometry results are expressed in terms of mean
fluorescence intensity ("MFI").
FIG. 3B is a graph depicting the results (expressed in terms of
MFI) of a flow cytometry study of serum plasma reactivity to B-CLL
cells (CD3-, CD19+) or T cells (CD3+, CD19-); serum plasma was
obtained from Patient B at the indicated periods pre-, peri-, or
post-alloHSCT.
FIG. 4A is a schematic diagram depicting the strategy used to
generate post-alloHSCT human Fab phage library.
FIG. 4B is a schematic diagram depicting the enrichment of the
phage library during selection rounds that included panning the
library on PBMC from untreated B-CLL patient .alpha..
FIG. 5A is a graph depicting, for each indicated selection round,
the output/input ratio of a Fab library phage.
FIG. 5B is a is a graph depicting, for each indicated selection
round, the results (absorbance at 405 nm) of a whole-cell ELISA
study probing Fab library phage reactivity to primary B-CLL
cells.
FIG. 6A is a series of four histogram panels depicting the results
(in terms of the number of events versus fluorescence intensity) of
flow cytometry studies testing JML-1 IgG1 reactivity to B-CLL PBMC
from Patient A, JML-1 IgG1 reactivity to B-CLL PBMC from Patient
.alpha., JML-1 IgG1 reactivity to CD19+ cells from healthy
volunteers, and JML-1 IgG1 reactivity to CD19- cells from healthy
volunteers, respectively.
FIG. 6B is a graph depicting the results of flow cytometry
FIG. 7 is a list of the amino acid sequences corresponding to the
variable region light chains (VL) of the following Fab: JML-1 (SEQ
ID NO:1) including the VL framework regions (FR1-FR4) (SEQ ID
NOs:2, 4, 6, and 8) and VL complementarity determining regions
(CDR1-CDR3) (SEQ ID NOs:3, 5, and 7) sequences therein; JML-3 (SEQ
ID NO:9) including the VL FR1-FR4 (SEQ ID NOs:10, 4, 11, and 8) and
VL CDR1-CDR3 (SEQ ID NOs:3, 5, and 7) sequences therein; JML-7 (SEQ
ID NO:12) including the VL FR1-FR4 (SEQ ID NOs:13, 4, 6, and 8) and
VL CDR1-CDR3 (SEQ ID NOs: 3, 5, and 7) sequences therein; and
JML-13 (SEQ ID NO:14) including the VL FR1-FR4 (SEQ ID NOs:15, 4,
17, and 8) and VL CDR1-CDR3 (SEQ ID NOs:3, 16, and 7) sequences
therein. Underlined residues indicate a difference relative to the
corresponding region in JML-1.
FIG. 8 is a list of the amino acid sequences corresponding to the
variable region heavy chains (VH) of the following Fab: JML-1 (SEQ
ID NO:18) including the VH FR1-FR4 (SEQ ID NOs:19, 21, 23, and 25)
and VH CDR1-CDR3 (SEQ ID NOs:20, 22, and 24) sequences therein;
JML-3 (SEQ ID NO:26) including the VH FR1-FR4 (SEQ ID NOs:27, 29,
23, and 25) and VH CDR1-CDR3 (SEQ ID NOs:28, 30, and 24) sequences
therein; JML-7 (SEQ ID NO:31) including the VH FR1-FR4 (SEQ ID
NOs:32, 29, 23, and 25) and VH CDR1-CDR3 (SEQ ID NOs:28, 30, and
24) sequences therein; and JML-13 (SEQ ID NO:33) including the VH
FR1-FR4 (SEQ ID NOs:34, 29, 23, and 25) and VH CDR1-CDR3 (SEQ ID
NOs:28, 30, and 24) sequences therein. Underlined residues indicate
a difference relative to the corresponding region in JML-1.
DETAILED DESCRIPTION OF THE INVENTION
The invention provides an isolated antibody with specificity for a
cell surface marker found on B-CLL and certain other CD19+ B cells.
Thus, the antibody is said to have "reactivity" or to be "reactive"
with such B cells. The invention also provides methods and
compositions thereof. Additionally the invention provides the
isolated B-CLL cell surface marker that is reactive with the
antibody of the invention.
In particular, the invention provides an antibody having B-CLL cell
surface reactivity comprising (a) a light chain variable domain
having at least 90% identity to a sequence selected from the group
consisting of SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:12, and SEQ ID
NO:14; (b) a heavy chain variable domain having at least 90%
identity to a sequence selected from the group consisting of SEQ ID
NO:18, SEQ ID NO:26, SEQ ID NO:31, or SEQ ID NO:33; or (c) both a
heavy chain of (a) and a light chain of (b). In a preferred
embodiment, the antibody comprises both a heavy chain of (a) and a
light chain of (b).
The antibody can be an isolated antibody having B-CLL cell surface
reactivity comprising a light chain variable domain having at least
90% identity to a sequence selected from the group consisting of
SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:12, and SEQ ID NO:14. In other
embodiments, the percentage identity can be at least 91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at
least 97%, at least 98%, or at least 99%, or even 100%. In
preferred embodiments, the light chain has at least 95% identity to
a sequence selected from the group consisting of SEQ ID NO:1, SEQ
ID NO:9, SEQ ID NO:12, and SEQ ID NO:14. In more preferred
embodiments, the light chain has 100% identity to a sequence
selected from the group consisting of SEQ ID NO:1, SEQ ID NO:9, SEQ
ID NO:12, and SEQ ID NO:14.
The antibody can be an isolated antibody having B-CLL cell surface
reactivity comprising a heavy chain variable domain having at least
90% identity to a sequence selected from the group consisting of
SEQ ID NO:18, SEQ ID NO:26, SEQ ID NO:31, and SEQ ID NO:33. In
other embodiments, the percentage identity can be at least 91%, at
least 92%, at least 93%, at least 94%, at least 95%, at least 96%,
at least 97%, at least 98%, or at least 99%, or even 100%. In
preferred embodiments, the light chain has at least 95% identity to
to a sequence selected from the group consisting of SEQ ID NO:18,
SEQ ID NO:26, SEQ ID NO:31, and SEQ ID NO:33. In more preferred
embodiments, the light chain has 100% identity to a sequence
selected from the group consisting of SEQ ID NO:18, SEQ ID NO:26,
SEQ ID NO:31, and SEQ ID NO:33.
In some embodiments, the antibody can comprise any heavy chain as
described above, in combination with any suitable light chain, such
as those described above. Likewise, the antibody can comprise any
of the light chains as described above in combination with any
suitable heavy chain, such as those described above. For example,
in certain embodiments, the antibody comprises (i) a light chain
variable domain with at least 90% identity to SEQ ID NO:1 and a
heavy chain variable domain with at least 90% identity to SEQ ID
NO:18, (ii) a light chain variable domain with at least 90%
identity to SEQ ID NO:9 and a heavy chain variable domain with at
least 90% identity to SEQ ID NO:26; (iii) a light chain variable
domain with at least 90% identity to SEQ ID NO:12 and a heavy chain
variable domain with at least 90% identity to SEQ ID NO:31; or (iv)
a light chain variable domain with at least 90% identity to SEQ ID
NO:14 a heavy chain variable domain with at least 90% identity to
SEQ ID NO:33.
In a preferred embodiment, the antibody comprises (i) the light
chain variable domain of SEQ ID NO:1 and the heavy chain variable
domain of SEQ ID NO:18, (ii) the light chain variable domain of SEQ
ID NO:9 and the heavy chain variable domain of SEQ ID NO:26; (iii)
the light chain variable domain of SEQ ID NO:12 and the heavy chain
variable domain of SEQ ID NO:31, or (iv) the light chain variable
domain of SEQ ID NO:14 and the heavy chain variable domain of SEQ
ID NO:33.
Percent (%) identity of peptide sequences can be calculated, for
example, as 100.times.(identical positions)/min(TG.sub.A,
TG.sub.B)], where TG.sub.A and TG.sub.B are the sum of the number
of residues and internal gap positions in peptide sequences A and B
in the alignment that minimizes TG.sub.A and TG.sub.B. See, e.g.,
Russell et al., J. Mol Biol., 244: 332-350 (1994).
The antibody of the invention can be any antibody including a full
length antibody or an antibody fragment. The antibody can be
monoclonal, recombinant, chimeric, or humanized. Preferably, the
antibody contains minimal or no potentially immunogenic animal
antibody-derived sequences. In a preferred embodiment, the antibody
is fully human. Furthermore, the antibody can be of any isotype
including without limitation IgA, IgD, IgE, IgG, or IgM. Thus, for
example, the antibody can be any IgA such as IgA1 or IgA2, or any
IgG such as IgG1, IgG2, IgG3, IgG4, or synthetic IgG. The antibody
can also be any antibody fragment having B-CLL cell surface
reactivity, such as a F(ab)2, Fv, scFv, IgG.DELTA.CH.sub.2,
F(ab')2, scFv2CH3, F(ab), VL, VH, scFv4, scFv3, scFv2, dsFv, Fv,
scFv-Fc, (scFv)2, a bispecific antibody such as a diabody, and a
bivalent antibody. The antibody can be any modified or synthetic
antibody, including, but not limited to, non-depleting IgG
antibodies, T-bodies, or other Fc or Fab variants of
antibodies.
In addition to a heavy chain as described above, the antibody of
the invention can further comprise a light chain selected from a
Fab library using sequential naive chain shuffling (e.g., as
described in International Patent Application Publication WO
2010/017103). Likewise, in addition to a light chain as described
above, the antibody of the invention can further comprise a heavy
chain selected from a Fab library using sequential naive chain
shuffling.
In some embodiments, the invention provides an isolated antibody
with B-CLL cell surface reactivity, comprising at least one CDR
having a sequence selected from the group consisting of SEQ ID NO:3
(VL CDR1), SEQ ID NO:5 (VL CDR2), SEQ ID NO:7 (VL CDR3), SEQ ID
NO:16 (VL CDR2), SEQ ID NO:20 (VH CDR1), SEQ ID NO:22 (VH CDR2),
SEQ ID NO:24 (VH CDR3), SEQ ID NO:28 (VH CDR1), and SEQ ID NO:30
(VH CDR2). The invention also provides an isolated antibody with
B-CLL cell surface reactivity comprising at least one or more
variants of the foregoing CDR sequences, which include 1, 2, or 3
substitutions, insertions, deletions, or combinations thereof in a
sequence selected from the group consisting of SEQ ID NO:3 (VL
CDR1), SEQ ID NO:5 (VL CDR2), SEQ ID NO:7 (VL CDR3), SEQ ID NO:16
(VL CDR2), SEQ ID NO:20 (VH CDR1), SEQ ID NO:22 (VH CDR2), SEQ ID
NO:24 (VH CDR3), SEQ ID NO:28 (VH CDR1), and SEQ ID NO:30 (VH
CDR2). For example, a recombinant chimeric or fully human antibody
(or fragment thereof) can include one, two, three, four, five, or
six of the foregoing CDR sequences.
The antibody of the invention can be produced by any suitable
technique, for example, using any suitable eukaryotic or
non-eukaryotic expression system. In certain embodiments, the
antibody is produced using a mammalian expression system.
The antibody of the invention can be produced using a suitable
non-eukaryotic expression system such as a bacterial expression
system. Bacterial expression systems can be used to produce
fragments such as a F(ab)2, Fv, scFv, IgG.DELTA.CH.sub.2, F(ab')2,
scFv2CH3, F(ab), VL, VH, scFv4, scFv3, scFv2, dsFv, Fv, scFv-Fc,
(scFv)2, and diabodies. Techniques for altering DNA coding
sequences to produce such fragments are known in the art.
The antibody of the invention can be conjugated to a synthetic
molecule using any type of suitable conjugation. Recombinant
engineering and incorporated selenocysteine (e.g., as described in
International Patent Application Publication WO 2008/122039) can be
used to conjugate a synthetic molecule. Other methods of
conjugation can include covalent coupling to native or engineered
lysine side-chain amines or cysteine side-chain thiols. See, e.g.,
Wu et al., Nat. Biotechnol., 23: 1137-1146 (2005). The synthetic
molecule can be any molecule such as one targeting a tumor. Of
course, it will be understood that the synthetic molecule also can
be a protein or an antibody.
Synthetic molecules include therapeutic agents such as cytotoxic,
cytostatic, or antiangiogenic agents and radioisotopes. A cytotoxic
agent can be a plant, fungal, or bacterial molecule (e.g., a
protein toxin). A therapeutic agent can be a maytansinoid (e.g.,
maytansinol or DM1 maytansinoid), a taxane, or a calicheamicin.
Therapeutic agents include vincristine and prednisone. A
therapeutic agent can be an antimetabolite (e.g., an antifolate
such as methotrexate, a fluoropyrimidine such as 5-fluorouracil,
cytosine arabinoside, or an analogue of purine or adenosine); an
intercalating agent (for example, an anthracycline such as
doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C,
dactinomycin, or mithramycin); a platinum derivative (e.g.,
cisplatin or carboplatin); an alkylating agent (e.g., nitrogen
mustard, melphalan, chlorambucil, busulphan, cyclophosphamide,
ifosfamide nitrosoureas, or thiotepa); an antimitotic agent (e.g.,
a vinca alkaloid like vincristine or taxoid such as paclitaxel or
docetaxel); a topoisomerase inhibitor (for example, etoposide, and
teniposide, amsacrine, or topotecan); a cell cycle inhibitor (for
example, a flavopyridol); or a microbtubule agent (e.g., an
epothilone, discodermolide analog, or eleutherobin analog). A
therapeutic agent can be a proteosome inhibitor or a topoisomerase
inhibitor such as bortezomib, amsacrine, etoposide, etoposide
phosphate, teniposide, or doxorubicin. Therapeutic radioisotopes
include yttrium (.sup.90Y), lutetium (.sup.177Lu), actinium
(.sup.225Ac), praseodymium, astatine (.sup.211At), rhenium
(.sup.186Re), bismuth (.sup.212Bi or .sup.213Bi), and rhodium
(.sup.188Rh). Antiangiogenic agents include linomide, bevacuzimab,
angiostatin, and razoxane. The synthetic molecule can be another
antibody such as rituximab or bevacuzimab.
A synthetic molecule can also be a label. Labels can be useful in
diagnostic applications and can include, for example, contrast
agents. A contrast agent can be a radioisotope label such as iodine
(.sup.131I or .sup.125I), indium (.sup.111In), technetium
(.sup.99Tc), phosphorus (.sup.32P), carbon (.sup.14C), tritium
(.sup.3H), other radioisotope (e.g., a radioactive ion), or a
therapeutic radioisotope listed above. Additionally, contrast
agents can include radiopaque materials, magnetic resonance imaging
(MRI) agents, ultrasound imaging agents, and any other contrast
agents suitable for detection by a device that images an animal
body. A synthetic molecule can also be a fluorescent label, a
biologically active enzyme label, a luminescent label, or a
chromophore label.
In some embodiments, the antibody can also have specificity for two
or more antigens on a CD19+ B-cell. For example, the antibody of
the invention can be engineered (e.g., as a bivalent diabody or a
conjugated Fab dimer or trimer) to have specificity for a second
CD19+ B-cell surface antigen, e.g., a second cell surface antigen
associated with B-CLL. In another embodiment, the antibody can be
engineered to have specificity for (in addition to its B-CLL cell
surface antigen) a second antigen that promotes activation or
targeting of cytotoxic effector cells.
The invention further provides eukaryotic or non-eukaryotic cells
that have been recombinantly engineered to produce an antibody of
the invention. The eukaryotic or non-eukaryotic cells can be used
as an expression system to produce the antibody of the invention.
In another embodiment, the invention provides B-CLL targeted immune
cells that are engineered to recombinantly express a B-CLL cell
surface reactive antibody of the invention. For example, the
invention provides a T-cell engineered to express an antibody of
the invention (e.g., an scFv, scFv-Fc, or (scFv)2), which is linked
to a synthetic molecule with the following domains: a spacer or
hinge region (e.g., a CD28, CD28, or IgG hinge), a transmembrane
region (e.g., a transmembrane canonical domain), and an
intracellular T-cell receptor (TCR) signaling domain, thereby
forming a T-body (or chimeric antigen receptor (CAR)).
Intracellular TCR signaling domains that can be included in a
T-body (or CAR) include, but are not limited to, CD3.xi.,
FcR-.gamma., and Syk-PTK signaling domains as well as the CD28,
4-1BB, and CD134 co-signaling domains. Methods for constructing
T-cells expressing a T-body (or CAR) are known in the art. See,
e.g., Marcu-Malina et al., Expert Opinion on Biological Therapy, 9:
539-564 (2009).
The invention provides a method of inhibiting the growth of CD19+ B
cells, e.g., B-CLL, that express a cell surface antigen for an
antibody of the invention. The method generally includes contacting
such B cells with an antibody of the invention. The antibody can be
a naked (unconjugated) antibody or an antibody conjugated to a
synthetic molecule, e.g., a cytotoxic, cytostatic, or
antiangiogenic agent or a radioisotope. The method can be used to
inhibit the CD19+ B cells in vitro or in a subject (i.e., in vivo).
The contacted B cells can be in, for example, a primary cell
culture or animal model of a disorder associated with such B cells,
e.g., a primary cell culture or animal model of B-CLL. The method
can be used, for example, to measure and/or rank (relative to
another antibody) the antibody's inhibitory activity for a specific
CD19+ B cell type. Inhibiting B cells can include blocking or
reducing the activity or growth of the B cells. Inhibiting can also
include the killing of the CD19+ B cells. While the method is not
bound by or limited to any mechanism of action, inhibitory activity
can be mediated by blocking signaling of the antibody's cell
surface antigen or an associated receptor. Inhibitory activity can
also be mediated by recruitment of immune system effectors to
attack the CD19+ B cells, e.g., by stimulating a CD8+ T-cell
response, complement-dependent cytotoxicity (CDC),
antibody-dependent cellular cytotoxicity (ADCC), or a combination
thereof.
The invention also provides a method of treating a subject that
has, is suspected to have, or is at risk for a disorder associated
with CD19+ B cells expressing a cell surface antigen for an
antibody of the invention. Generally, the method includes
administering a therapeutically effective amount of an isolated
antibody of the invention to the subject. The antibody can be any
of the invention as described above. Thus, the antibody can be
chimeric, humanized, synthetic, F(ab)2, Fv, scFv,
IgG.DELTA.CH.sub.2, F(ab')2, scFv2CH3, F(ab), VL, VH, scFv4, scFv3,
scFv2, dsFv, Fv, or (scFv)2. In some embodiments, the method
includes administering an IgG, an scFv, a dsFv, a F(ab').sub.2, a
diabody, or a bivalent antibody. Preferably the antibody is a fully
human antibody. The administered antibody can be conjugated to a
synthetic molecule described above, e.g., a cytotoxic, cytostatic,
or antiangiogenic agent or a therapeutic radioisotope. An exemplary
cytotoxic agent is Pseudomonas exotoxin A (PE38). Disorders that
can be treated include, for example, B-CLL.
The invention also provides a method of treating a subject that
has, is suspected to have, or is at risk for a disorder associated
with elevated levels of CD19+ B cells (e.g., B-CLL) by adoptive
transfer of the genetically engineered T-cells described herein,
which express an antibody of the invention as a T-body (or CAR).
Recombinant technology can be used to introduce T-body (or CAR)
encoding genetic material into any suitable T-cells, e.g., central
memory T-cells from the subject to be treated. The T-cells carrying
the genetic material can be expanded (e.g., in the presence of
cytokines). The genetically engineered T-cells are transferred,
typically by infusion, to the patient. The transferred T-cells of
the invention can then mount an immune response against the CD19+ B
cells in the subject. The adoptive transfer method can be used, for
example, to treat subjects that have or are suspected to have
B-CLL.
In some embodiments, the foregoing methods of treatment can further
include co-administering a second therapeutic agent for the
disorder associated with elevated levels of CD19+ B cells. For
example, when the disorder to be treated involves B cell lymphoma
or leukemia (e.g., B-CLL), the method can further include
co-administration of a cytotoxic, cystostatic, or antiangiogenic
agent suitable for treating the B cell cancer such as, e.g.,
co-administration of rituximab or alemtuzumab or utilization of a
CHOP chemotherapeutic regimen.
The terms "treat," "treating," "treatment," and "therapeutically
effective" used herein do not necessarily imply 100% or complete
treatment. Rather, there are varying degrees of treatment, which
one of ordinary skill in the art recognizes as having a potential
benefit or therapeutic effect. In this respect, the inventive
method can provide any amount of any level of treatment.
Furthermore, the treatment provided by the inventive method can
include the treatment of one or more conditions or symptoms of the
disease being treated.
In another embodiment, the invention provides method of detecting
in a test sample an altered level of CD19+ B cells (e.g., B-CLL
cells) that are reactive with the antibody of the invention.
Generally, the method includes contacting a test sample with an
antibody of the invention and evaluating (e.g, deteimining) the
amount of antibody that selectively binds to cells in the sample
(e.g., by reference to a control level) to thereby qualitatively or
quantitatively determine the level of CD19+ B cells (e.g., B-CLL
cells) reactive with the antibody of the invention. A test sample
can be from a cell culture or from a test subject, e.g., a plasma
or a tissue sample from a subject that has, is suspected to have,
or is at risk for a disease or condition associated with an altered
level of such CD19+ B cells. A control level desirably corresponds
to the amount of the antibody detected when the same antibody is
contacted to a corresponding sample(s) from one or more control
cultures or subjects. Methods of using the antibody of the
invention to determine altered levels of CD19+ B cells can include
any immunoassay such as immuno- (Western) blotting, enzyme-linked
immunosorbent assay (ELISA), and flow cytometry, e.g.,
fluorescence-activated cell sorting (FACS) analysis.
The method of detection can be used to screen for the presence of a
disorder (e.g., B-CLL) associated with an altered level of CD19+ B
cells reactive with the antibody of the invention. The method
includes obtaining a sample from a test subject in need of
screening, e.g., a subject that has, is suspected to have, or is at
risk for such a disorder. The level of reactive CD19+ B cells in
the sample is measured using an antibody of the invention, and the
level in the sample is compared to a control level. The control
level represents, for example, the mean level (e.g., the amount or
concentration) in sample(s) from one or, preferably, multiple
control group subjects that do not have a disorder associated with
the reactive CD19+ B cells (e.g., control group subjects that do
not have B-CLL). Alternatively, the control level can correspond to
the level or mean level of reactive CD19+ B cells in one or more
samples taken from the test subject at one or more prior times,
when the test subject did not have, or did not exhibit, a condition
associated with an altered level of the reactive CD19+ B cells. A
significantly altered (e.g., higher) level of the reactive CD19+ B
cells in the test sample relative to the control level is
indicative of a disorder associated with the reactive CD19+ B cells
(e.g. B-CLL) in the subject.
Additionally, the method of detection can be used to monitor the
progress of a disorder, such as B-CLL, associated with an elevated
level of CD19+ B cells reactive with the antibody of the invention.
The method includes obtaining a sample from a subject in need of
screening, e.g., a subject having been diagnosed or suspected to
have a disorder associated with an altered level of the reactive
B-cells. The level of reactive B-cells in the sample is measured
using an antibody of the invention, and the level in the sample is
compared to a control level corresponding to the level or mean
level of reactive B-cells in one or more samples taken from the
test subject at one or more prior times. Levels of reactive B-cells
(e.g., B-CLL) that are significantly elevated or decreased relative
to the control level indicate that the subject's disorder is
deteriorating or improving, respectively.
The invention provides a method for screening a subject for an
altered level of CD19+ B cells that are reactive with the antibody
of the invention. Generally, the method includes administering to
the subject an antibody of the invention that is conjugated to a
label (e.g., a contrast agent), imaging the subject in a manner
suitable for detecting the label, and determining whether a region
in the subject has an altered density or concentration of label as
compared to the background level of label in proximal tissue.
Alternatively, the method includes determining whether there is an
altered density or concentration of label in a region as compared
to the density or concentration of label previously detected in the
same region of the subject. Methods of imaging a subject can
include x-ray imaging, x-ray computed tomography (CT) imaging
(e.g., CT angiography (CTA) imaging), magnetic resonance (MR)
imaging, magnetic resonance angiography (MRA), nuclear medicine,
ultrasound (US) imaging, optical imaging, elastography, infrared
imaging, microwave imaging, and the like, as appropriate for
detecting the label conjugated to the antibody. In a preferred
embodiment, the subject has, is suspected to have, or is at risk
for a reactive B cell tumor, such as B-CLL, and the method is used
to detect the presence or absence of the tumor. In another
embodiment, the method can be used to monitor the size or density
of a reactive B cell tumor, such as B-CLL, over time, e.g., during
a course of treatment.
The invention also provides a composition (e.g., a pharmaceutical
composition) comprising an antibody as described above and a
carrier (e.g., a pharmaceutically acceptable carrier). Suitable
compositions, such as pharmaceutical compositions, can be prepared
from any of the antibodies described herein. An exemplary
composition includes a carrier and a fully human antibody having
(i) the light chain variable domain of SEQ ID NO:1 and/or the heavy
chain variable domain of SEQ ID NO:18, (ii) the light chain
variable domain of SEQ ID NO:9 and/or the heavy chain variable
domain of SEQ ID NO:26; (iii) the light chain variable domain of
SEQ ID NO:12 and/or the heavy chain variable domain of SEQ ID
NO:31, or (iv) the light chain variable domain of SEQ ID NO:14
and/or the heavy chain variable domain of SEQ ID NO:33. Another
exemplary composition comprises a carrier and a fully human or
humanized antibody having one, two, three, four, five, or six CDRs
selected from the group consisting of SEQ ID NO:3 (VL CDR1), SEQ ID
NO:5 (VL CDR2), SEQ ID NO:7 (VL CDR3), SEQ ID NO:16 (VL CDR2), SEQ
ID NO:20 (VH CDR1), SEQ ID NO:22 (VH CDR2), SEQ ID NO:24 (VH CDR3),
SEQ ID NO:28 (VH CDR1), and SEQ ID NO:30 (VH CDR2).
The composition of the invention comprises a carrier for the
antibody, desirably a pharmaceutically acceptable carrier. The
pharmaceutically acceptable carrier can be any suitable
pharmaceutically acceptable carrier. The term "pharmaceutically
acceptable carrier" as used herein means one or more compatible
solid or liquid fillers, diluents, other excipients, or
encapsulating substances which are suitable for administration into
a human or veterinary patient (e.g., a physiologically acceptable
carrier or a pharmacologically acceptable carrier). The term
"carrier" denotes an organic or inorganic ingredient, natural or
synthetic, with which the active ingredient is combined to
facilitate the use of the active ingredient. The pharmaceutically
acceptable carrier can be co-mingled with one or more of the active
components, e.g., a hybrid molecule, and with each other, when more
than one pharmaceutically acceptable carrier is present in the
composition, in a manner so as not to substantially impair the
desired pharmaceutical efficacy. "Pharmaceutically acceptable"
materials typically are capable of administration to a patient
without the production of significant undesirable physiological
effects such as nausea, dizziness, rash, or gastric upset. It is,
for example, desirable for a composition comprising a
pharmaceutically acceptable carrier not to be immunogenic when
administered to a human patient for therapeutic purposes.
The pharmaceutical composition can contain suitable buffering
agents, including, for example, acetic acid in a salt, citric acid
in a salt, boric acid in a salt, and phosphoric acid in a salt. The
pharmaceutical compositions also optionally can contain suitable
preservatives, such as benzalkonium chloride, chlorobutanol,
parabens, and thimerosal.
The pharmaceutical composition can be presented in unit dosage form
and can be prepared by any suitable method, many of which are well
known in the art of pharmacy. Such methods include the step of
bringing the antibody of the invention into association with a
carrier that constitutes one or more accessory ingredients. In
general, the composition is prepared by uniformly and intimately
bringing the active agent into association with a liquid carrier, a
finely divided solid carrier, or both, and then, if necessary,
shaping the product.
A composition suitable for parenteral administration conveniently
comprises a sterile aqueous preparation of the inventive
composition, which preferably is isotonic with the blood of the
recipient. This aqueous preparation can be formulated according to
known methods using suitable dispersing or wetting agents and
suspending agents. The sterile injectable preparation also can be a
sterile injectable solution or suspension in a non-toxic
parenterally-acceptable diluent or solvent, for example, as a
solution in 1,3-butane diol. Among the acceptable vehicles and
solvents that can be employed are water, Ringer's solution, and
isotonic sodium chloride solution. In addition, sterile, fixed oils
are conventionally employed as a solvent or suspending medium. For
this purpose any bland fixed oil can be employed, such as synthetic
mono-or di-glycerides. In addition, fatty acids such as oleic acid
can be used in the preparation of injectables. Carrier formulations
suitable for oral, subcutaneous, intravenous, intramuscular, etc.
administrations can be found in Remington's Pharmaceutical
Sciences, Mack Publishing Co., Easton, Pa.
The delivery systems useful in the context of the invention include
time-released, delayed release, and sustained release delivery
systems. The inventive composition can be used in conjunction with
other therapeutic agents or therapies. Such systems can avoid
repeated administrations of the inventive composition, thereby
increasing convenience to the subject and the physician, and may be
particularly suitable for certain compositions of the
invention.
Many types of release delivery systems are available and known to
those of ordinary skill in the art. Suitable release delivery
systems include polymer base systems such as
poly(lactide-glycolide), copolyoxalates, polycaprolactones,
polyesteramides, polyorthoesters, polyhydroxybutyric acid, and
polyanhydrides. Microcapsules of the foregoing polymers containing
drugs are described in, for example, U.S. Pat. No. 5,075,109.
Delivery systems also include non-polymer systems that are lipids
including sterols such as cholesterol, cholesterol esters, and
fatty acids or neutral fats such as mono- di- and tri-glycerides;
hydrogel release systems; sylastic systems; peptide based systems;
wax coatings; compressed tablets using conventional binders and
excipients; partially fused implants; and the like. Specific
examples include, but are not limited to: (a) erosional systems in
which the active composition is contained in a form within a matrix
such as described in U.S. Pat. Nos. 4,452,775, 4,667,014,
4,748,034, and 5,239,660 and (b) diffusional systems in which an
active component permeates at a controlled rate from a polymer such
as described in U.S. Pat. Nos. 3,832,253 and 3,854,480. In
addition, pump-based hardware delivery systems can be used, some of
which are adapted for implantation.
The term "subject" is used herein, for example, in connection with
therapeutic and diagnostic methods, to refer to human or animal
subjects (e.g., mammals). Animal subjects include, but are not
limited to, animal models, such as, mammalian models of conditions
or disorders associated with an altered level of CD19+ B cells that
are reactive with the antibody of the invention, e.g., B-CLL.
The invention also provides kits suitable for carrying out the
methods of the invention. Typically, a kit comprises two or more
components required for performing a therapeutic or detection
method of the invention. Kit components include, but are not
limited to, one or more antibodies of the invention, appropriate
reagents, and/or equipment.
A kit can comprise an antibody of the invention and an immunoassay
buffer suitable for detecting CD19+ B cells that are reactive with
the antibody of the invention (e.g. by whole-cell ELISA or FACS).
The kit may also contain one or more microtiter plates, standards,
assay diluents, wash buffers, adhesive plate covers, and/or
instructions for carrying out a method of the invention using the
kit. The kit can include an antibody of the invention bound to a
substrate (e.g., a multi-well plate or a chip), which is suitably
packaged and useful to detect CD19+ B cells (e.g., B-CLL cells)
that are reactive with the antibody of the invention. In some
embodiments, the kit includes an antibody of the invention that is
conjugated to a label, such as a fluorescent label, a biologically
active enzyme label, a luminescent label, or a chromophore label.
The kit can further include reagents for visualizing the conjugated
antibody, e.g., a substrate for the enzyme. In some embodiments,
the kit includes an antibody of the invention that is conjugated to
a contrast agent and, optionally, one or more reagents or pieces of
equipment useful for imaging the antibody in a subject.
Generally the antibody of the invention in a kit is suitably
packaged, e.g., in a vial, pouch, ampoule, and/or any container
appropriate for a therapeutic or detection method. Kit components
can be provided as concentrates (including lyophilized
compositions), which may be further diluted prior to use, or the
kit components can be provided at the concentration intended for
use. When the antibody of the invention is intended to be used in
vivo, single dosages may be provided in sterilized containers
having the desired amount and concentration of agents.
In another embodiment, the invention provides the isolated B-CLL
cell surface marker that is reactive with the antibody of the
invention. The B-CLL cell surface marker can be isolated from
primary B-CLL cells using the antibody of the invention in
conjunction with art-known techniques such as immunoprecipitation.
The isolated B-CLL cell surface marker can be used, for example, to
generate additional B-CLL cell surface reactive antibodies. Thus,
the isolated B-CLL cell surface marker can be used to immunize a
suitable animal (e.g., a goat, rabbit, mouse, etc.) to thereby
generate polyclonal antibody with specificity for B-CLL. An
antibody producing cell can be isolated from such an animal and
immortalized to thereby generate monoclonal antibodies with
specificity for B-CLL. Monoclonal antibodies can be further
engineered to generate chimeric or fully human antibodies. Such
antibodies, which are reactive with B-CLL cell surface, can be used
in the methods described herein.
The isolated B-CLL cell surface marker can also be used to identify
other therapeutic or diagnostic compounds (e.g., small molecule
compounds, cytotoxins, etc.) that bind to the marker. Techniques
for identifying such binding compounds include screening analytical
microarrays or one-bead-one-compound (OBOC) libraries for
immobilized compounds that bind to the isolated marker.
Alternatively, the isolated B-CLL surface marker or the antibody of
the invention can be immobilized on a substrate (e.g., a
microarray) and used to screen compound libraries for those that
inhibit the B-CLL surface marker binding to the antibody of the
invention. Thus, the invention provides a method for identifying
therapeutic or diagnostic compounds that includes isolating the
B-CLL cell surface marker, e.g., using the antibody of the
invention, and using the isolated B-CLL cell surface marker to
identify a compound that binds to the marker and/or inhibits the
marker from binding to the antibody of the invention.
The following examples further illustrate the invention but, of
course, should not be construed as in any way limiting its
scope.
EXAMPLE 1
This example demonstrates a sensitive flow cytometry assay for
detecting serum antibodies with B-CLL cell surface reactivity in
patients following alloHSCT.
Qdot 655 nanocrystals (Quantum Dot Corporation, Hayward, Calif.)
were conjugated to goat F(ab').sub.2 anti-human IgG polyclonal
antibody (g.alpha.h-Qdot) for use as a secondary antibody.
Multiparameter flow cytometry was performed using an LSR II
instrument (BD Biosciences, Immunocytometry Systems, San Jose,
Calif.). Plasma samples from B-CLL patients and alloHSCT donors
were prepared from blood and stored at -80.degree. C. Peripheral
blood mononuclear cells (PBMC) were prepared from blood using
lymphocyte separation medium (MP Biomedicals, Solon, Ohio) and
cryopreserved until use.
In a pilot experiment, approximately 5.times.10.sup.5 PBMC were
prepared from an untreated B-CLL patient. Detection was initially
complicated by B-CLL cell surface expression of IgM, IgD, and Fcy
receptors such as CD32B, as shown in FIG. 1. The flow cytometry
results are depicted in FIG. 2, panel A, which indicated that PBMC
were reactive against g.alpha.h-Qdot secondary antibody alone.
To avoid g.alpha.h-Qdot detection of the human Ig displayed on
B-CLL cell surface, PBMC were first blocked with 4% (v/v) normal
goat serum in phosphate buffered saline (PBS) (which was also used
for all subsequent dilutions and washes) followed by 100 .mu.g/mL
unconjugated goat Fab anti-human IgG polyclonal antibodies (Jackson
ImmunoResearch Laboratories, West Grove, Pa.). All incubation steps
were done on ice for 1 hour. After two washes, cells were incubated
with 1 .mu.g/mL rituximab (chimeric mouse/human anti-human CD20 mAb
with human IgG1.kappa. constant domains from Genentech, South San
Francisco, Calif., and Biogen Idec, Cambridge, Mass.) or
alemtuzumab (humanized anti-human CD52 mAb with human IgG1.kappa.
constant domains from Genzyme, Cambridge, Mass.). Following two
washes, the cells were incubated with 20 nM of g.alpha.h-Qdot
secondary antibody. The cells were also stained with
CD3-FITC/CD19-PE or CD5-FITC/CD19-PE SIMULTEST reagents (BD
Biosciences) for gating T cell and B cells, and propidium iodide
for excluding dead cells from the analysis. After two more washes,
a total of 20,000 gated events were collected for each sample in a
list mode file, and data analysis was performed using FACS Convert
and CELLQUEST software (BD Biosciences). The pilot flow cytometry
assay results are depicted in FIG. 2 and demonstrate that blocking
largely eliminated g.alpha.h-Qdot secondary antibody reactivity
(see panel B) and allowed for specific detection of B-CLL cell
surface antigens using rituximab as primary antibody (see panel C).
This flow cytometry assay was similarly effective for specifically
detecting alemtuzumab B-CLL antigen.
The foregoing flow cytometry assay (with blocking) was used to
probe PBMC from two alloHSCT-treated B-CLL patients prior to
induction chemotherapy. CD3-FITC/CD19-PE two-color SIMULTEST.TM.
reagents were used for gating PBMC into B cells (CD3-CD19+,
dominated by B-CLL cells) and T cells (CD3+CD19-). Instead of
rituximab or alemtuzumab, the primary antibody used in these
experiments was a 1:2 dilution of human plasma samples obtained
from patients at various times before, during, and after alloHSCT.
Plasma samples obtained from donors or pooled human AB serum
(Invitrogen, Carlsbad, Calif.) were used as primary antibody
negative controls. Characteristics of the two B-CLL patients
(Patients A and B) are set forth in Table 1.
TABLE-US-00001 TABLE 1 Patient A Patient B Indication Relapse
Relapse Sex Male Female Age at 52 48 Enrollment Enrollment 2003
2002 National Insti- NCT00055744 NCT00003838 tutes of Health
Clinical Trial Web Site Identifier Induction EPOCH + FR (etoposide,
FC (fludarabine & Chemotherapy prednisone, cyclophospha-
cyclophosphamide) mide, & doxorubicin) + (fludarabine,
rituximab, & vincristine) Donor (HLA- Brother Brother matched,
6/6; A, B, and DR) GVHD CSP + MTX (cyclosporine + CSP + MTX
Prophylaxis methotrexate) Acute GVHD No No Chronic GVHD No No
Response Partial response; complete Complete response response
after DLI Status - Molecular remission Molecular remission May
2009
FIGS. 3A and 3B depict flow cytometry assay results as mean
fluorescence intensity ("MFI") for the B cells (black bars) and T
cells (white bars) obtained from Patients A and B at the various
time points before (pre), at (peri), and after (post) alloHSCT. The
results shown in FIG. 3A indicate that post-alloHSCT plasma from
Patient A contained substantial, though transient, B-CLL cell
surface reactivity, which peaked six months after transplantation.
A weaker T cell reactivity that correlated with B-CLL cell surface
reactivity was also observed in Patient A (see FIG. 3A). The
results in FIG. 3B show a similar pattern for Patient B, although
Patient B transient B-CLL cell surface reactivity peaked at ten
months after transplantation and was not accompanied by T cell
surface reactivity (see FIG. 3B). By contrast, the pre- and
peri-alloHSCT plasma from Patients A and B, plasma from the
alloHSCT donor of Patient A, and pooled control plasma from healthy
volunteers were all negative for B or T cell reactivity.
Despite the limited availability of post-alloHSCT plasma from
Patients A and B at the defined time points, reactive post-alloHSCT
serum antibodies were analyzed for cell surface reactivity with
autologous B-CLL cells, autologous T cells, allogeneic B cells, and
third party B cells using the flow cytometry assay described above.
For Patient A, allogeneic B cells were derived from Patient A PBMC
collected fifteen months after transplantation. For Patient B,
allogeneic B cells were derived from donor PBMC. Rituximab and
alemtuzumab were also analyzed for comparison. Table 2 indicates
the flow cytometry results for each class of cells in terms of an
MFI ratio (sample over background). An MFI ratio <2 is "-"; MFI
ratio >2 and <5 is "weak", MFI ratio >5 and <20 is "+",
and <100, is "++", and MFI ratio >100 is "+++". The results
in Table 2 indicate some cell surface reactivity with allogeneic B
cells and third party B cells. Additional analyses with secondary
antibodies specific for human Ig isotypes suggested that both IgG
and IgM contributed to the transient B-CLL cell surface
reactivity.
TABLE-US-00002 TABLE 2 Allo- Third Autologous Autologous geneic
party B-CLL cells T cells B cells B cells Patient A ++ + - weak
post 6 m plasma Patient B ++ weak ++ + post 10 m plasma rituximab
++ (Patient A) - (Patient A) ++ ++ + (Patient B) - (Patient B)
alemtuzumab +++ (Patient A) - (Patient A) +++ +++ +++ (Patient B) -
(Patient B)
When compared to clinical data, the peak in transient B-CLL cell
surface reactivity approximately paralleled the time points at
which (a) full donor chimerism was achieved and (b) the
disappearance of B-CLL cells by flow cytometry and PCR was noted.
This suggested that the observed serum antibody response against
B-CLL cells is an antigen-dependent phenomenon, likely involving
both autologous and allogeneic epitopes of cell surface
antigens.
The foregoing results demonstrate the successful development of an
assay with sufficient sensitivity and suitability to detect serum
antibodies with B-CLL cell surface reactivity in alloHSCT
recipients. The foregoing data also indicate the temporal and
immunological profile for these anti-B-CLL serum antibodies.
EXAMPLE 2
This example demonstrates the generation of a post-alloHSCT human
Fab library.
A human Fab library was generated using cryopreserved post-alloHSCT
PBMC collected from Patient A at the peak of serum antibody
response, i.e., at six months after transplantation, as described
in Example 1. Total RNA was extracted from 2.5.times.10.sup.7 PBMC
using TRI Reagent (Molecular Research Center, Cincinnati, Ohio) and
further purified using the RNEASY.TM. Mini Kit from Qiagen
(Germantown, Md.). Approximately 100 .mu.g total RNA was isolated
and validated by agarose gel electrophoresis. First-strand cDNA
synthesis from total RNA using an oligo(dT) primer and
SUPERSCRIPT.TM. reverse transcriptase (Invitrogen) was performed
according to the manufacturer's protocol. V.sub..kappa.,
V.sub..lamda., and V.sub.H encoding sequences were separately
amplified from first-strand cDNA by a 35-cycle PCR using the
FastStart High Fidelity PCR System from Roche (Indianapolis, Ind.)
and combinations of 12 sense/1 antisense primers for V.sub..kappa.,
20 sense/3 antisense primers for V.sub..lamda., and 19 sense/6
antisense primers for V.sub.H, for a total of 186 different
combinations, encompassing all human germlines. The antisense
primers for V.sub..lamda., and V.sub.H align to J.sub..lamda. and
J.sub.H germlines, respectively, whereas the antisense primer for
V.sub..kappa. aligns to the C.sub..kappa. encoding sequence. Human
C.sub..LAMBDA.-pelB and C.sub..lamda.-pelB encoding sequences
required for the V.sub..kappa.-C.sub..kappa.-V.sub.H and
V.sub..lamda.-C.sub..lamda.-V.sub.H cassette assembly,
respectively, were amplified from pC.sub..kappa. and pC.sub..lamda.
as described in Hofer et al., J. Immunol. Methods, 318: 75-87
(2007), and Kwong et al., J. Mol. Biol., 384: 1143-1156 (2008),
respectively. V.sub..kappa.-C.sub..kappa.-V.sub.H and
V.sub..lamda.-C.sub..lamda.-V.sub.H cassettes were assembled in one
fusion step based on 3-fragment overlap extension PCR, digested
with SfiI, and cloned into pC3C as also described in Kwong et al.
(2008), supra. Transformation of E. coli strain XL1-Blue
(Stratagene) by electroporation yielded 9.8.times.10.sup.7 and
1.6.times.10.sup.8 independent transformants for the .kappa. and
.lamda. phagemid libraries, respectively. Randomly picked
independent transformants from each library were analyzed for Fab
expression by ELISA and for sequence diversity by DNA
fingerprinting using AluI as described in Popkov et al., J. Mol.
Biol., 325: 325-335 (2003). Using VCSM13 helper phage (Stratagene),
the pooled phagemid library was converted to a phage library as
described Barbas et al., Phage Display: A Laboratory Manual, Cold
Spring Harbor Laboratory Press (Cold Spring Harbor, N.Y., 2001).
The phage library was stored at 4.degree. C. after adding sodium
azide to a final concentration of 0.02% (w/v).
FIG. 4A schematically depicts how the phage library was generated.
Despite the depleted and not yet fully recovered B cell repertoire
in post-alloHSCT PBMC, the post-alloHSCT PBMC phage library
included 2.6.times.10.sup.8 independent Fab clones and represented
71% for all primer combinations. This was closer than had been
expected to the 89% success rate for primer combinations found in a
library generated from the normal PBMC of a healthy volunteer.
Table 3 sets forth the successful primer combination rate for each
amplified variable region from the post-alloHSCT or normal PBMC.
Phage library integrity and diversity was confirmed by ELISA and
DNA fingerprinting of unselected Fab clones.
TABLE-US-00003 TABLE 3 Post-alloHSCT PBMC Normal PBMC Successful
primer 10/12 (83%) 12/12 (100%) combinations V.sub..kappa.
Successful primer 41/60 (68%) 59/60 (98%) combinations
V.sub..lamda. Successful primer 81/114 (71%) 95/114 (83%)
combinations V.sub.H Total of successful 132/186 (71%) 166/186
(89%) primer combinations Phagemid pC3C not applicable Library size
2.6 .times. 10.sup.8 not applicable E. coli strain XL1-Blue not
applicable Helper phase VCSM13 not applicable
The foregoing results demonstrate the high complexity and integrity
of the post-alloHSCT human Fab library that was generated.
EXAMPLE 3
This example demonstrates the enrichment of Fab with B-CLL surface
reactivity from the post-alloHSCT human Fab library.
The post-alloHSCT human Fab library was selected on cryopreserved
PBMC (consisting of >85% B-CLL cells) from an untreated B-CLL
patient (Patient .alpha.). The cells were maintained in 6-well
tissue culture plates for 1-2 days in RPMI 1640 medium (Invitrogen)
supplemented with 5% (v/v) autologous serum. Five rounds of panning
were carried out using the phage display protocol described in
Barbas et al., Phage Display: A Laboratory Manual, Cold Spring
Harbor Laboratory Press (Cold Spring Harbor, N.Y., 2001), and Rader
et al., Methods Mol. Biol., 525: 101-128 (2009). All incubations
were at room temperature unless noted otherwise. In the first
round, freshly re-amplified phage library of Example 2 was
pre-selected for functional Fab display through panning on rat
anti-hemagglutinin (HA) mAb (Roche), which was immobilized on three
wells of a 96-well ELISA plate (Costar 3690; Corning, Corning,
N.Y.) at 500 ng/well. In the second round, 0.5 mL of fresh phage
was first mixed with 0.5 mL of 5% (v/v) autologous serum in PBS and
0.5 mL of 1% (w/v) bovine serum albumin ("BSA") in PBS. After
adding sodium azide to a final concentration of 0.12% (w/v), the
phage were incubated for 30 minutes. Primary B-CLL cells from one
6-well tissue culture plate were harvested, collected through
lymphocyte separation medium (MP Biomedicals), resuspended in 1.5
mL of 5% (v/v) autologous serum in PBS, counted
(4.2.times.10.sup.7), added to the 1.5 mL phage preparation in a 15
mL polypropylene tube, and incubated for 30 minutes with gentle
agitation every 5 minutes. After washing three times with 15 mL
PBS, the cells were resuspended in 0.6 mL PBS containing 10 mg/mL
trypsin, shaken at 37.degree. C. and 250 rpm for 30 minutes, and
added to two 2-mL XL1-Blue cultures, resuming the phage display
protocol. The third round was identical to the second round, except
that 1.2.times.10.sup.7 primary B-CLL cells were used. In the
fourth round, selection for functional Fab display was repeated
using two wells with immobilized rat anti-HA mAb at 200 ng/well.
The fifth round was identical to the second and third round, except
that 5.times.10.sup.7 primary B-CLL cells were used and four washes
with 15 mL PBS were carried out.
In sum, the post-alloHSCT human Fab library was enriched by three
selection rounds (i.e., rounds 2, 3, and 5) that included panning
the library on PBMC from untreated B-CLL Patient .alpha., as
schematically depicted in FIG. 4B. Two additional rounds of panning
on immobilized rat anti-HA mAb (i.e., rounds 1 and 4) were carried
out prior to the first and after the second PBMC panning round to
eliminate phage that did not display functional Fab with HA
tag.
The enrichment of phage displaying Fab with B-CLL cell surface
reactivity in selection rounds 2, 3, and 5 was monitored by phage
output-to-input ratio titering as described in Barbas et al. (2001)
and Rader et al. (2009), supra. The phage output-to-input ratio for
each selection round is depicted in FIG. 5A. The phage
output-to-input ratio increased approximately 50-fold over the
three cell panning selection rounds.
Polyclonal phage from selection rounds 2, 3, and 5 were diluted to
approximately 1.times.10.sup.11 phage in 75 .mu.L PBS and stored on
ice. All subsequent steps were carried out in a V-bottom 96-well
tissue culture plate (Costar 3894) (Corning, Lowell, Mass.) at room
temperature using PBS for washing and dilution. Approximately
5.times.10.sup.5 primary B-CLL cells from Patient 60 were washed
twice, resuspended in the 75-.mu.L phage preparations or in PBS as
a negative control, and incubated for 1 hour on a rocker.
Subsequently, the cells were washed twice and incubated with 100
.mu.L of a 1:1,000 dilution of mouse anti-phage mAb conjugated to
horse radish peroxidase ("HRP") (GE Healthcare, Piscataway, N.J.)
for 1 hour. The cells then were washed twice, resuspended in 50
.mu.L HRP substrate solution, and incubated for 20 minutes. See
Rader et al. (2009), supra. Absorbance at 405 nm was determined
with an ELISA plate reader. The absorbance signal for each
selection round was adjusted by subtracting the negative control
(i.e., cells and detection antibody alone) signal. The adjusted
absorbance signals for rounds 2, 3, and 5 are depicted in FIG. 5B
and confirm a progressive increase of phage displaying human Fab
with cell surface reactivity.
The foregoing results indicate that Fab with B-CLL surface
reactivity were successfully enriched by panning against B-CLL
cells from untreated Patient .alpha..
EXAMPLE 4
This example demonstrates the identification and characterization
of post-alloHSCT human Fab library clones having B-CLL surface
reactivity.
One hundred selected Fab clones from the last selection round in
Example 3 were analyzed for Fab expression by ELISA and for
sequence diversity using AluI DNA fingerprinting as described in
Popkov et al., J. Mol. Biol., 325: 325-335 (2003). Of these, 85
clones were found to have Fab expression, B-CLL cell surface
reactivity, and readable DNA fingerprints. Among these 85 Fab
clones, 73 belonged to one of seven repeated DNA fingerprints, and
63 belonged to one of four dominating patterns with eight or more
apparently identical Fab clones. Representative Fab clones,
designated JML-1, -3, -7, and -13, each from one of the four
dominating patterns, were further analyzed by DNA sequencing. The
deduced amino acid sequences of JML-1, -3, -7, and -13 light chain
and heavy chain variable domains are depicted in FIGS. 7 and 8,
respectively.
The amino acid sequences of the four variable domains were analyzed
with respect to germline origin, LCDR3 and HCDR3 sequences, and
overall sequence identity to each other. The results of these
sequence analyses are shown in Table 4. The human germline
analysis, which was based on DNA alignments generated by
IMGT/V-QUEST sequence analysis software (available at the
INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM web site),
indicated that all four Fab clones were highly homologous with
identical V.sub..kappa., J.sub..kappa., D.sub.H, and J.sub.H
germline origins and identical LCDR3 and HCDR3 sequences. However,
all Fab clones differed by at least four amino acid mutations, and
JML-1 had a different V.sub.H germline origin than JML-3, -7, and
-13, despite their identical HCDR3 sequences. A BLAST search of
protein databases at the National Center for Biotechnology
Information web site revealed that this HCDR3 sequence, GGQTIDI, is
unique among rearranged heavy chains.
TABLE-US-00004 TABLE 4 JML-1 JML-3 JML-7 JML-13 Light chain Human
germlines V.sub..kappa. 1-39 V.sub..kappa. 1-39 V.sub..kappa. 1-39
V.sub..kappa. 1-39 J.sub..kappa. 3 J.sub..kappa. 3 J.sub..kappa. 3
J.sub..kappa. 3 Deviation from V.sub..kappa. germline 0/95 2/95
1/95 3/95 LCDR3 sequence QQSYSTPFT QQSYSTPFT QQSYSTPFT QQSYSTPFT
(SEQ ID NO: 7) (SEQ ID NO: 7) (SEQ ID NO: 7) (SEQ ID NO: 7)
Sequence identity to JML-1 100% 98% 99% 97% Sequence identity to
JML-3 98% 100% 97% 95% Sequence identity to JML-7 99% 97% 100% 96%
Sequence identity to JML-13 97% 95% 96% 100% Heavy chain Human
germlines V.sub.H 3-9 V.sub.H 3-30 V.sub.H 3-30 V.sub.H 3-30
D.sub.H 3-10 D.sub.H 3-10 D.sub.H 3-10 D.sub.H 3-10 J.sub.H 3
J.sub.H 3 J.sub.H 3 J.sub.H 3 Deviation from V.sub.H germline 7/98
2/98 2/98 1/98 HCDR3 sequence GGQTIDI GGQTIDI GGQTIDI GGQTIDI (SEQ
ID NO: 24) (SEQ ID NO: 24) (SEQ ID NO: 24) (SEQ ID NO: 24) Sequence
identity to JML-1 100% 89% 90% 88% Sequence identity to JML-3 89%
100% 99% 99% Sequence identity to JML-7 90% 99% 100% 98% Sequence
identity to JML-13 88% 99% 98% 100%
As indicated in Table 4, JML-3, -7, and -13 included approximately
2% overall deviation from their V.sub..kappa. and V.sub.H germline
sequences. JML-ldeviated approximately 7% from its V.sub.H germline
sequence (see Table 4). This deviation is evidence of somatic
hypermutation in the heavy chain of JML-1.
Four to five selected clones with identical fingerprints
corresponding to JML-1, -3, -7, or -13 were selected, and Fab
expression was induced with isopropyl
.beta.-D-1-thiogalactopyranoside as described in Rader et al.
(2009), supra. Supernatants were pooled and concentrated ten-fold
using a 15 mL Amicon Ultra Centrifugal Filter Device with 10-kDa
molecular weight cut off (Millipore, Billerica, Mass.) to generate
crude Fab samples corresponding to JML-1, -3, -7, and -13. Crude
Fab samples (75 .mu.L) were analyzed by whole-cell ELISA, as
described in Example 3, using PBMC from untreated B-CLL Patient
.alpha.. Crude Fab samples prepared from pooled clones with the
JML-1 fingerprint consistently revealed the strongest B-CLL cell
surface reactivity.
The foregoing results demonstrates the preparation of four
exemplary Fab antibodies of the invention. The high homologies
among JML-1, -3, -7, and -13 Fab, in particular their identical
LCDR3 and HCDR3 sequences, imply that these four exemplary Fab
antibodies recognize the same antigen. However, their primary
usefulness is based on B-CLL cell surface reactivity and does not
require that they recognize the same surface antigen.
EXAMPLE 5
This example demonstrates the generation, expression, purification,
and biotinylation of JML-1 IgG 1.
JML-1 V.sub.H and light chain encoding sequences were PCR amplified
using appropriately designed primers and cloned into mammalian
expression vector PIGG as previously described in Hofer et al., J.
Immunol. Methods, 318: 75-87 (2007) and Rader et al., FASEB J., 16:
2000-2002 (2002). 300 .mu.g of the resulting PIGG-JML-1 plasmid in
293FECTIN transfection reagent (Invitrogen) was transiently
transfected into 3.times.10.sup.8 HEK 293F cells. Transfected cells
were maintained in 300 mL FreeStyle serum-free medium in a 500-mL
spinner flask on a stirring platform at 75 rpm (CELLSPIN System;
Integra Biosciences, Chur, Switzerland) in a humidified atmosphere
containing 8% CO.sub.2 at 37.degree. C. After four days, the medium
was collected after centrifugation, replaced for additional three
to four days, and collected again. Pooled supernatants were then
processed, and IgG1 was purified using a 1 mL recombinant Protein A
HITRAP column (GE Healthcare) as previously described in Hofer et
al. (2007), supra. The quality and quantity of purified IgG1 was
determined by SDS-PAGE and A.sub.280 absorbance. In parallel with
JML-1, the previously described human anti-tetanus toxoid mAb TT11
IgG1 was expressed and purified as a negative control. See Kwong et
al., J. Mol. Biol., 384: 1143-1156 (2008). Purified JML-1 IgG1 and
TT11 IgG1 were biotinylated using the BiotinTag Micro-Biotinylation
Kit (Sigma-Aldrich, St. Louis, Mo.). The number of conjugated
biotin molecules per IgG1 molecule was approximately four for each
of JML-1 and TT11 IgG1.
The flow cytometry assay described in Example 1 was used to confirm
that JML-1 IgG1, but not TT11 IgG1, strongly bound to B-CLL cells
from Patient .alpha. (who was used for library selection). JML-1
IgG1 also recognized B-CLL cells from Patient A (the alloHSCT
recipient from whom the library had been generated). The results of
the foregoing flow cytometry assays are depicted in the histograms
labeled Patient .alpha. and Patient A, respectively, in FIG.
6A.
To characterize JML-1 IgG, PBMC were prepared from 12 B-CLL
patients that were not involved in generating and selecting the Fab
library. PBMC were also prepared from the freshly drawn blood of
healthy volunteers, and the CD19+ and CD19- subpopulations were
purified by magnetic activated cell sorting (MACS) using CD19
MicroBeads (Miltenyi Biotec, Auburn, Calif.). Additionally JML-1
IgG was characterized for its ability to bind the following eleven
cell lines. Five cell lines were generated using Epstein-Barr virus
(EBV) to transforin B lymphoblastoid cell lines (EBV-LCL) obtained
from PBMC of healthy volunteers (583, 0745, and 1363) and B-CLL
patients (18-7-3 and 18-1-12) as previously described. Aman et al.,
J. Exp. Med., 159: 208-220 (1984). PCR amplification of Ig heavy
chain VDJ gene fragments from genomic DNA indicated that EBV-LCL
0745 was polyclonal and that EBV-LCL 18-7-3 and 18-1-12 were
monoclonal, albeit different in HCDR3 lengths than the
corresponding B-CLL cells (suggesting EBV transformation of normal
B cells present in these B-CLL patients). The B-CLL cell line EHEB
was obtained from the German Collection of Microorganisms and Cell
Cultures (Braunschweig, Germany). Saltman et al., Leuk. Res., 14:
381-387 (1990). The B-CLL cell line 232-B4 was kindly provided by
Dr. Anders Rosen (Linkoping University, Linkoping, Sweden).
Wendel-Hansen et al., Leukemia, 8: 476-484 (1994). Human Burkitt's
lymphoma B-cell lines Daudi, Raji, and Ramos and human mantle cell
lymphoma B-cell line JeKo-1 were obtained from American Type
Culture Collection (Manassas, Va.).
Approximately 5.times.10.sup.5 cells (from the PBMC of a B-CLL
patient, from MACS-separated PBMC of a healthy volunteer, or from a
cell line) were blocked with pooled human AB serum. All incubation
steps were done on ice for 1 hour. The cells were incubated with 10
.mu.g/mL biotinylated JML-1 or biotinylated TT11 IgG1 in 2% (v/v)
fetal bovine serum (FBS) (Hyclone, Logan, Utah) in PBS (which was
used for all subsequent dilutions and washes), followed by two
washes, incubation with 2 .mu.g/mL PE-coupled streptavidin (BD
Biosciences), and two more washes. Propidium iodide was added to
exclude dead cells from the analysis. Flow cytometry was performed
using a FACSCALIBUR.TM.instrument (BD Biosciences) and analyzed
with CELLQUEST.TM. software. The flow cytometry assay results for
each of the foregoing cell sources are depicted as mean
fluorescence intensity ("MFI") in FIG. 5B.
As shown in FIG. 5B, JML-1 IgG1 but not TT11 IgG1 was reactive to
B-CLL in 11 of the 12 B-CLL patients that were not involved in
generating or selecting the Fab library. These results indicate
that the antigen recognized by JML-1 is broadly expressed in B-CLL
and is not restricted to Patients A and .alpha..
Some variability of JML-1 IgG1 reactivity was found among the cells
derived from different B-CLL patients and in the CD19+
subpopulation of PBMC derived from different healthy volunteers
(but not in the corresponding CD19- subpopulations), as depicted in
FIGS. 5A and 5B. Nonetheless, compared to primary B cells, the mean
JML-1 IgG1 reactivity measured for primary B-CLL cells was
significantly higher (p=0.023) as indicated in FIG. 5B. None of the
eleven human B-cell lines analyzed revealed significant JML-1 IgG1
reactivity (see FIG. 5B). Collectively, these findings suggested
that the antigen recognized by JML-1 IgG is restricted to primary B
cells and over-expressed in primary B-CLL cells.
The foregoing results demonstrate the preparation of an IgG
antibody of the invention and its usefulness for detecting B-CLL
and other CD19+ B cells.
All references, including publications, patent applications, and
patents, cited herein are hereby incorporated by reference to the
same extent as if each reference were individually and specifically
indicated to be incorporated by reference and were set forth in its
entirety herein.
The use of the terms "a" and "an" and "the" and similar referents
in the context of describing the invention (especially in the
context of the following claims) are to be construed to cover both
the singular and the plural, unless otherwise indicated herein or
clearly contradicted by context. The terms "comprising," "having,"
"including," and "containing" are to be construed as open-ended
terms (i.e., meaning "including, but not limited to,") unless
otherwise noted. Recitation of ranges of values herein are merely
intended to serve as a shorthand method of referring individually
to each separate value falling within the range, unless otherwise
indicated herein, and each separate value is incorporated into the
specification as if it were individually recited herein. All
methods described herein can be performed in any suitable order
unless otherwise indicated herein or otherwise clearly contradicted
by context. The use of any and all examples, or exemplary language
(e.g., "such as") provided herein, is intended merely to better
illuminate the invention and does not pose a limitation on the
scope of the invention unless otherwise claimed. No language in the
specification should be construed as indicating any non-claimed
element as essential to the practice of the invention.
Preferred embodiments of this invention are described herein,
including the best mode known to the inventors for carrying out the
invention. Variations of those preferred embodiments may become
apparent to those of ordinary skill in the art upon reading the
foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
SEQUENCE LISTINGS
1
341107PRTArtificial SequenceSource phage library of human antibody
sequences 1Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile
Ser Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Ser Tyr Ser Thr Pro Phe 85 90 95Thr Phe Gly Pro Gly Thr Lys
Val Asp Ile Lys 100 105223PRTArtificial SequenceSource phage
library of human antibody sequences 2Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys 20311PRTArtificial SequenceSource phage library of human
antibody sequences 3Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn1 5
10415PRTArtificial SequenceSource phage library of human antibody
sequences 4Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
Tyr1 5 10 1557PRTArtificial SequenceSource phage library of human
antibody sequences 5Ala Ala Ser Ser Leu Gln Ser1 5632PRTArtificial
SequenceSource phage library of human antibody sequences 6Gly Val
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr1 5 10 15Leu
Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 20 25
3079PRTArtificial SequenceSource phage library of human antibody
sequences 7Gln Gln Ser Tyr Ser Thr Pro Phe Thr1 5810PRTArtificial
SequenceSource phage library of human antibody sequences 8Phe Gly
Pro Gly Thr Lys Val Asp Ile Lys1 5 109107PRTArtificial
SequenceSource phage library of human antibody sequences 9Asp Ile
Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25
30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr
Ser Thr Pro Phe 85 90 95Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys
100 1051023PRTArtificial SequenceSource phage library of human
antibody sequences 10Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
201132PRTArtificial SequenceSource phage library of human antibody
sequences 11Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr1 5 10 15Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr
Tyr Tyr Cys 20 25 3012107PRTArtificial SequenceSource phage library
of human antibody sequences 12Asp Ile Gln Leu Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu
Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Phe 85 90 95Thr Phe
Gly Pro Gly Thr Lys Val Asp Ile Lys 100 1051323PRTArtificial
SequenceSource phage library of human antibody sequences 13Asp Ile
Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp
Arg Val Thr Ile Thr Cys 2014107PRTArtificial SequenceSource phage
library of human antibody sequences 14Asp Ile Gln Met Thr Gln Ser
Pro Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Gly Ala Ser
Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Phe 85 90
95Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys 100
1051523PRTArtificial SequenceSource phage library of human antibody
sequences 15Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys 20167PRTArtificial
SequenceSource phage library of human antibody sequences 16Gly Ala
Ser Ser Leu Gln Ser1 51732PRTArtificial SequenceSource phage
library of human antibody sequences 17Gly Val Pro Ser Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr1 5 10 15Leu Thr Ile Thr Ser Leu
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 20 25 3018116PRTArtificial
SequenceSource phage library of human antibody sequences 18Lys Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25
30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Gly Gly Gln Thr Ile Asp Ile Trp Gly
Gln Gly Thr Met Val 100 105 110Thr Val Ser Ser 1151930PRTArtificial
SequenceSource phage library of human antibody sequences 19Lys Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp 20 25
30205PRTArtificial SequenceSource phage library of human antibody
sequences 20Asp Tyr Gly Met His1 52114PRTArtificial SequenceSource
phage library of human antibody sequences 21Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val Ser1 5 102217PRTArtificial
SequenceSource phage library of human antibody sequences 22Gly Ile
Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val Lys1 5 10
15Gly2332PRTArtificial SequenceSource phage library of human
antibody sequences 23Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr Leu Gln1 5 10 15Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys Ala Arg 20 25 30247PRTArtificial SequenceSource
phage library of human antibody sequences 24Gly Gly Gln Thr Ile Asp
Ile1 52511PRTArtificial SequenceSource phage library of human
antibody sequences 25Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser1 5
1026116PRTArtificial SequenceSource phage library of human antibody
sequences 26Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr
Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Gly Gln Thr Ile
Asp Ile Trp Gly Gln Gly Thr Met Val 100 105 110Thr Val Ser Ser
1152730PRTArtificial SequenceSource phage library of human antibody
sequences 27Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Val Val Gln Pro
Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser 20 25 30285PRTArtificial SequenceSource phage library of human
antibody sequences 28Ser Tyr Gly Met His1 52914PRTArtificial
SequenceSource phage library of human antibody sequences 29Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala1 5
103017PRTArtificial SequenceSource phage library of human antibody
sequences 30Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser
Val Lys1 5 10 15Gly31116PRTArtificial SequenceSource phage library
of human antibody sequences 31Lys Val Gln Leu Leu Glu Ser Gly Gly
Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Ser Tyr Asp
Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Gly Gly Gln Thr Ile Asp Ile Trp Gly Gln Gly Thr Met Val 100 105
110Thr Val Ser Ser 1153230PRTArtificial SequenceSource phage
library of human antibody sequences 32Lys Val Gln Leu Leu Glu Ser
Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser 20 25 3033116PRTArtificial
SequenceSource phage library of human antibody sequences 33Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Gly Gly Gln Thr Ile Asp Ile Trp Gly
Gln Gly Thr Met Val 100 105 110Thr Val Ser Ser 1153430PRTArtificial
SequenceSource phage library of human antibody sequences 34Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30
* * * * *